A wastewater-based epidemiological study of licit and illicit drugs among schoolchildren and students in Slovenia

Ivona Krizman-Matasić1, Taja Verovšek2,3, Ada Hočevar Grom4, Urška Blaznik4, Andreja Drev4 and Ester Heath2,3

1 Rudjer Boškovic Institute, Bijenicka 54, Zagreb, Croatia2Jožef Stefan Institute, Jamova 39, Ljubljana, Slovenia3Jožef Stefan International Postgraduate School, Jamova 39, Ljubljana, Slovenia4National Institute of Public Health, Trubarjeva 2, Ljubljana, Slovenia

Wastewater samples (n=42):• 20 primary schools,

15 secondary schools,*12 centres for higher education.

• 37 institutions in urban area, 10 institutions in rural area.

THE AIM OF THE STUDY

Application of wastewater-based epidemiology (WBE) to different Slovenian educational bodies to:• Investigate licit and illicit drug consumption trends based on:

• Institutions’ educational level: primary schools, secondary schools, centres for higher education• Geographic location: seven Slovenian municipalities• Level of urbanity: urban, rural

• Compare results obtained by our study and by traditionally used epidemiological methods (European School Survey Project on Alcohol and Other Drugs (ESPAD), Health Behaviour in School-Aged Children (HBSC) survey)

PARTICIPATION

METHOD AND RESULT HIGHLIGHTS

Table 1 List of selected licit and illicit drugs and their biomarkers (n=19)

Fig. 1 Number of samples obtained per municipality Fig. 2 Number of samples regarding institutions’ educational level per municipality

RESULTS

General findings

• Commonly detected biomarkers: nicotine, cannabis, alcohol, cocaine (COC and BE)

• None detected: heroin biomarker (6-AM), methadone biomarkers (MTHD, EDDP)

• Biomarkers per sample: 4 to 12 (modus: 7)

REFERENCES ACKNOWLEDGEMENTS

Spatial variation Variation based on educational level

Comparison of the findings: WBE – traditional epidemiological methods

• Urban areas have higher detection rates (exceptions: MAMP, AMP)

• Biomarkers detected per sample: similar for urban and rural areas (modus: 7)

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Fig. 5 Detection rate (%) of biomarkers in samples from urban and rural areas

* 2 schools offering secondary and higher education

Trends in urban and rural areas

• Most commonly detected biomarkers: nicotine, cannabis, alcohol

• Other commonly detected biomarkers: COC (Cities: 1,3, 4, 7), MOR (Cities: 2, 5 ,6), MDMA (City 4), COD (City 2)

• Stimulants: AMP (only in City 1), MAMP (in Cities: 1, 4, 5, 6), MDMA (in Cities: 1, 7)

• Biomarkers per sample: highest in City 1 (modus: 11)

• Most commonly detected biomarkers: nicotine, cannabis, cocaine (COC)

• Most commonly detected biomarkers in samples of centres for higher education: BE, MOR

• Detection rates of biomarkers increase with the level of education

• Lower number of biomarkers detected in samples from primary schools than in samples from secondary schools and centres for higher education

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Fig. 4 Detection rate (%) of biomarkers in different municipalities

• Sample selection: headmasters/deans approval, evaluation of sampling possibility• Sampling campaign: Tuesday/Wednesday/Thursday, over class period – one 7-h composite wastewater sample

per school• Sampling difficulties: Physical boundaries of the sewer - some samples (n=5) contain wastewater from more than

one wastewater point-source• Analysis1,2,3: filtration, extraction and pre-concentration of analytes (illicit drugs) or direct injection (nicotine and

alcohol biomarkers), separation and detection of selected urinary biomarkers (n=19) by LC-MS/MS

Drug Selected biomarker(s)

Basic drugs

Cocaine Cocaine (COC), benzoylecgonine (BE), cocaethylene (COE)

Amphetamine Amphetamine (AMP)

Methamphetamine Methamphetamine (MAMP)

Ecstasy 3,4-methylenedioxymethamphetamine (MDMA)

Heroin Morphine (MOR), morphine-3-glucuronide (M3G), 6-acetylmorphine (6-AM)

Codeine Codeine (COD)

Methadone Methadone (MTHD), 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP)

Cannabinoids

THC 11-Nor-9-carboxy-THC (THC-COOH), 1-Hydroxy-THC (THC-OH)

Tobacco

Nicotine Nicotine (NIC), cotinine (COT), trans-3‘-hydroxycotinine (HCOT)

Alcohol

Ethanol Ethyl sulphate (EtS), ethyl glucuronide (EtG)

• Results of WBE agree with the latest traditional epidemiological study - European School Survey Project on Alcohol and Other Drugs (ESPAD) and the Health Behaviour in School-Aged Children (HBSC) project

• Licit drugs: the highest consumption was found to be consumption of licit drugs (tobacco, alcohol)

• Illicit drugs: cannabis is the most common illicit drug

• Stimulants: cocaine the most prevalent

Sampling and wastewater analysis

[1] I. Senta, I. Krizman, M. Ahel, et al. Anal Bioanal Chem. 2013, 405: 3255. https://doi.org/10.1007/s00216-013-6720-9[2] F. Y. Lai, C. Gartner, W. Hall, et al. Addiction. 2018, 113: 1127–1136. doi:10.1111/add.14157[3] T. Rodríguez-Álvarez, R. Rodil, R. Cela, et al. J. Chromatogr. A. 2014, 1328:35– 42. http://dx.doi.org/10.1016/j.chroma.2013.12.076

Research was supported by ARRS Program group P1-0143 and applicative project L1-9191

DECLARATION OF INTERESTS

No conflict of interest was reported by the authors.

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Fig. 3 Detection rate (%) of biomarkers in obtained samples